Antibiotic resistance among Gram-negative bacteria such as Escherichia coli, Klebsiella species, and other Enterobacteriaceae is emerging worldwide at an alarming rate due to the misuse and overuse of antibiotics (Queenan & Bush (2007) Clin. Microbiol. Rev. 20: 440-458; Richard et al., (1994) J. Infect. Dis. 170: 377-383; Spellberg et al., (2013) N. Engl. J. Med. 368: 299-302). Particularly, the resistance against broad-spectrum β-lactam antibiotics has become a major public health concern. Of many antibiotics, carbapenems have the broadest spectrum of activity and greatest potency against different types of bacteria, and have become the “last resort” in treatment of serious bacterial infections (Bradley et al., (1999) Int. J. Antimicrob. Agents 11: 93-100; Papp-Wallace et al., (2011) Antimicrob. Agents Chemother. 55: 4943-4960). However, Carbapenem-Resistant Enterobacteriaceaes (CREs) are now frequently observed and the number of cases is steadily growing, mostly due to acquired carbapenemases (Queenan & Bush (2007) Clin. Microbiol. Rev. 20: 440-458; Nordmann et al., (2011) Emerg. Infect. Dis. 17: 1791-1798).
Carbapenemases are a group of β-lactamases with the ability to hydrolyze almost all β-lactam antibiotics and include Ambler class A β-lactamases of the KPC-type, the metallo-β-lactamases (MBLs) (Ambler class B) of VIM-, IMP-, New Delhi metallo-β-lactamase (NDM), and the relatively rare class D carbapenemase of OXA-48 type (Queenan & Bush (2007) Clin. Microbiol. Rev. 20: 440-458; Nordmann et al., (2011) Emerg. Infect. Dis. 17: 1791-1798; Miriagou et al., (2010) Clin. Microbiol. Infect. 16: 112-122). Carbapenemases are also increasingly reported as the cause of therapeutic failures in hospital- and community-acquired infections, especially for MBLs, because of its transmission between different strains and poor susceptibility to clinically available inhibitors (Queenan & Bush (2007) Clin. Microbiol. Rev. 20: 440-458; Walsh et al., (2011) Clin. Microbiol. 49: 3222-3227). Therefore, rapid and accurate detection of MBL-producers is critically important for appropriate antibacterial chemotherapies and rigorous infection control.
Currently, the standard diagnostic methods for the detection of β-lactamases, such as the Modified Hodge Test (MHT) (Nordmann et al., (2012) Clin. Microbiol. Infect. 18: 432-438; Cohen & Leverstein-van (2010) Int. J. Antimicrob. Agents 36: 205-210; Bernabeu et al., (2012) Diagn. Microbiol. Infect. Dis. 74: 88-90; Dortet et al., (2012) Antimicrob. Agents. Chemother. 56: 6437-6440) and “double-disk synergy test” (DDST) (Arakawa et al., (2000) J. Clin. Microbiol. 38: 40-43) lack the necessary specificity and sensitivity, and are time-consuming, typically requiring at least 24-48 hours for accurate detection of most bacteria, but days for those bacteria with a slow growth rate.
PCR-based methods (Poirel et al., (2011) Diagn. Microbiol. Infect. Dis. 70: 119-125; Avlami et al., (2010) J. Microbiol. Methods. 83: 185-187; Chen et al., (2011) J. Clin. Microbiol. 49: 579-585) and mass spectrometry (Burckhardt & Zimmermann (2011) J. Clin. Microbiol. 49: 3321-3324; Hrabak et al., (2011) J. Clin. Microbiol. 49: 3327-3332) have high accuracy and sensitivity, but are associated with the disadvantages of high cost, heavy instrument requirements, and inability to detect newly-evolved carbapenemase genes. The Carba-NP test (CNP) recently developed by Nordmann (Nordmann et al., (2012) Emerg. Infect. Dis. 18: 1503-1507; Dortet et al., (2012) J. Clin. Microbiol. 50: 3773-3776; Dortet et al., (2012) Antimicrob. Agents Chemother. 56: 6437-6440) has high specificity, but it is only applicable to a bacterial lysate.
In contrast, fluorescence-based bioanalytical assays offer high sensitivity, ease of use, rapid detection, low cost, and little or no need for biological sample processing before analysis. A number of fluorescent probes that enable the detection of the β-lactamase activity with high sensitivity have been developed (Gao et al., (2003) J. Am. Chem. Soc. 125: 11146-11147; Zlokarnic et al., (1998) Science 279: 86-88; Xing et al., (2005) J. Am. Chem. Soc. 127: 4158-4159; Kong et al., (2010) Proc. Natl. Acad. Sci. USA 419: 9-16; Xie et al., (2012) Nat. Chem. 4: 802-809; Watanabe et al., (2010) Bioconjugate Chem. 21: 2320-2326; Zhang et al., (2012) Angew. Chem. Int. Ed. 51: 1865-1868; Yang et al., (2007) J. Am. Chem. Soc. 129: 266-267). However, most of these probes lack specificity for carbapenemases. Some probes have been used for detecting carbapenemases only when combined with certain inhibitors (Zhang et al., (2012) Angew. Chem. Int. Ed. 51: 1865-1868). Thus, few fluorescent probes are available for specifically detecting carbapenemases. The present disclosure provides a series of fluorogenic probes based on cephalosporin with a 6,7-trans configuration suitable for the specific detection of MBLs in bacteria.